When immunohistochemistry is difficult

For some beginner students, the immunohistochemistry step is more cumbersome, one step wrong and one step wrong.
There are also many experiment partners to make fun of the small make up, obviously feel to do according to the conditions of the teachers, but it is not to get the expected result.
Look at this today!

• 1.Comparison of the selection of paraffin sections and frozen sections

• 2.An anti-selection and technique

(1) selection of monoclonal and polyclonal antibodies.
The inherent characteristics of monoclonal and polyclonal antibodies determine their advantages and limitations in IHC/ICC.
Monoclonal antibodies are produced by single B cell clones, which represent homogenous groups and can be combined with single epitopes with high affinity.
This is especially useful when testing a member of a protein family, which has a high proportion of amino acids homologous.

Antibody binding often relies on target protein to maintain its natural state of conception.
Interaction with other proteins, post-translational modification, temperature, pH, fixation and salt concentration all affect the proximity of the antibody to the target table.
Polyclonal antibodies are heterogeneous to identify multiple table bits, so they are less affected by changes in the protein conformation.

In general, polyclonal antibodies are more stable than monoclonal antibodies in the range of certain pH and salt concentrations.
For these reasons, polyclonal antibodies are more commonly used in IHC/ICC experiments.

(2) selection of application scope.
Some of them can only be used for Western Blotting, or immunohistochemistry, immunofluorescence, immune precipitation, etc.
Even a paraffin section or frozen section.

(3) the selection of species reactivity.
This is important to show that there may be a species difference in the antibody, which is suitable for detecting the antigens in the animal.

(4) genera source, the average rabbit source is polyclone;
In mice, many are monoclonal, but there are others.
Choose the corresponding two reactance according to this source.

(5) selection of manufacturers.

• 3.The principle of selection of closed serum

(1) there are residual sites on the membrane or slice that can be nonspecific adsorbed antibodies, resulting in false positives for subsequent results.

(2) serum is usually of the resistance to the same source and the closed, the animal's own antibody in serum, in advance can cross reaction sites and organization are combined, or in a later step, if the and two combination will cause the background.

(3) it can also be used in calf serum, BSA, and sheep serum, but not in accordance with one source.

• 4.The selection, frequency and timing of the washing method on PBS

(1) wash separately and prevent cross-reaction from causing pollution.

Antibodies used in the cases of clinical testing every day a lot, types and project too much, if in a fight after the incubation, wash them in a tank, it will cause the cross contamination, influence the final result.
The correct way to do this is to do the washing separately, and wash the PBS to ensure that the slices have no chance of cross-contamination.

(2) gently rinse to prevent the peeling of sections.

Wash slices, remove slices, on the PBS from flushing, gently let PBS flowing down from the top down, don't pick up the biopsy aim the PBS wash, due to rush out of the PBS has a certain impact, so it is easy to make slice surrounding cause loose, lead to slice off.

(3) the washing time should be sufficient to thoroughly wash the combination of substances.

It is a waste of time, and the second is that it can easily lead to the shedding of the slices.
Sliced flushing, according to the practice thought, is washed with a small beaker softly on the slices can be thoroughly flushed to not combined with material, without other additional conditions, rinse well on the slices injected PBS, last 2 min or so enough.

• 5.Why should a fight 4 ℃ after incubation for temperature of 37 ℃ after?

(1) on the one hand, prevent the slice from being directly put into PBS easy strip from 4.

(2) on the other hand, the binding of antigen antibody is more stable.
Generally don't need, but with weak expression of antigen is likely to be useful, 4 DHS and 37 C DHS C molecular motion in a different way, the former molecular collision probability and speed is less than the latter, the latter with faster, but the sensitivity is improved and easy to cause nonspecific staining.

6.How are immunohistochemical results analyzed

(1) positive staining cell counting method.
In the 40 * photoscope, randomly selected 10 different fields of view, artificial or machine count positive colouring cells, 3-6 different animal tissues of each group, and then compared between groups.

(2) grey density analysis method.
The gray-density analysis was performed with image j by selecting the same region and the same conditions in different groups and different animal tissues, and then statistical analysis was done.

(3) scoring method.
Through the biopsy under optical microscope respectively according to the degree of dyeing (0-3 negative staining, light yellow, light brown, dark brown), scope of positive rate (1-4:0-25%, 26-50%, 51-76, 76-75%), could eventually scores, then the comparison.

For the above methods, there are pros and cons, please choose carefully.
The only way to get the right result is to make a very high quality slice that is evenly colored and very light in the background.

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